Evolution of human tuberculosis: a systematic review and meta-analysis of paleopathological evidence.

Tuberculosis is a re-emerging disease and is a major problem in both developing and developed countries today. An estimated one third of the world's population is infected and almost two million people die from the disease each year. Bone lesions occur in 3-5% of active tuberculosis cases and can be used to diagnose the disease in ancient skeletal remains. A meta-analysis was conducted on 531 palaeopathological tuberculosis cases from 221 sites (7250 BCE to 1899) on all continents for the purpose of testing two hypotheses; (1) the frequency of bone lesions does not change through time and (2) the distribution of lesions throughout the skeleton does not change over time. The frequency of bone lesions was found to significantly decrease over time (P<0.05). The distribution of bone lesions was found to change from mainly spinal in earlier time periods to include more cases in other regions of the skeleton (long bones, joints, hands, feet) in later time periods. This difference in distribution was evaluated using a Chi-squared test and found to be significant (P<0.01). These findings are an important addition to the current knowledge of the evolution of the disease and the Mycobacterium tuberculosis.

Combining near infrared spectroscopy and multivariate analysis as a tool to differentiate different strains of Saccharomyces cerevisiae: a metabolomic study.

Near-infrared (NIR) spectroscopy has gained wide acceptance within the food and agriculture industries as a rapid analytical tool. NIR spectroscopy offers the advantage of rapid, non-destructive analysis and routine operation is simple and opens the possibility of using spectra to obtain the 'fingerprint' of a sample. The aim of this study was to explore the potential of combining visible (VIS) and near-infrared (NIR) spectroscopy, together with multivariate analysis, in establishing the function of genes, by investigating the metabolic profiles produced by Saccharomyces cerevisiae deletion strains sourced from the EUROSCARF yeast collection. Spectra (400-2500 nm) were acquired with a FOSS NIRSystems6500 (Foss NIRSystems), in transmittance mode. Principal component analysis (PCA) and linear discriminant analysis (LDA) were used in order to visualize graphically the relative differences and similarities of yeast deletion strains. VIS and NIR spectroscopy showed great promise as a screening tool for both discriminating between yeast strains and grouping strains with deletions in genes that disturb similar metabolic pathways. These results indicate that the methods may be useful in defining the function of genes that produce no obvious phenotype.

The use of anonymous DNA markers in assessing worldwide relatedness in the yeast species Pichia kluyveri Bedford and Kudrjavzev.

Pichia kluyveri, a sexual ascomycetous yeast from cactus necroses and acidic fruit, is divided into three varieties. We used physiological, RAPD, and AFLP data to compare 46 P. kluyveri strains collected worldwide to investigate relationships among varieties. Physiology did not place all strains into described varieties. Although the combined AFLP and RAPD data produced a single most parsimonious tree, separate analysis of AFLP and RAPD data resulted in significantly different trees (by the partition homogeneity test). We then compared the distribution of strains per band to an expected distribution. This suggested we could separate both the AFLP and RAPD datasets into bands from rapidly and slowly changing DNA regions. When only bands from slowly changing regions (from each dataset) were included in the analysis, both the RAPD and AFLP datasets supported a single tree. This second tree did not differ significantly from the cladogram based on all of the DNA data, which we accepted as the best estimate of the phylogeny of these yeast strains. Based on this phylogeny, we were able to demonstrate the strong influence of geography on the population structure of this yeast, confirm the monophyly of one variety, question the utility of maintaining another variety, and demonstrate that the physiological differences used to separate the varieties did not do so in all cases.

The incidence of killer activity of non-Saccharomyces yeasts towards indigenous yeast species of grape must: potential application in wine fermentation.

Fourteen killer yeasts were assayed for their ability to kill species of yeast that are commonly associated with fermenting grape must and wine. A total of 147 of a possible 364 killer-sensitive interactions were observed at pH 4.5. Of the killer yeasts studied, Pichia anomala NCYC 434 displayed the broadest killing range. At a pH value comparable with those of wine ferments, pH 3.5, the incidence of killer-sensitive interactions was reduced by 700% across all the yeasts. Williopsis saturnus var. mrakii CBS 1707 exhibited the broadest killing range at the lower pH, killing more than half of the tester strains. Intraspecific variation in sensitivity to killer yeasts was observed in all species where more than one strain was tested. Also, in strains of Pichia anomala, Kluyveromyces lactis and Pichia membranifaciens, the three species in which more than one killer yeast was analysed, intraspecific variation in killer activity was observed.

AFLP fingerprinting for analysis of yeast genetic variation.

Amplified fragment length polymorphism (AFLP) was used to investigate genetic variation in commercial strains, type strains and winery isolates from a number of yeast species. AFLP was shown to be effective in discriminating closely related strains. Furthermore, sufficient similarity in the fingerprints produced by yeasts of a given species allowed classification of unknown isolates. The applicability of the method for determining genome similarities between yeasts was investigated by performing cluster analysis on the AFLP data. Results from two species, Saccharomyces cerevisiae and Dekkera bruxellensis, illustrate that AFLP is useful for the study of intraspecific genetic relatedness. The value of the technique in strain differentiation, species identification and the analysis of genetic similarity demonstrates the potential of AFLP in yeast ecology and evolutionary studies.

Differentiation and species identification of yeasts using PCR.

A PCR-based method has been developed that permits both intraspecies differentiation and species identification of yeast isolates. Oligonucleotide primers that are complementary to intron splice sites were used to produce PCR fingerprints that display polymorphisms between different species of indigenous wine yeasts. Although polymorphisms existed between isolates of the same species, the banding patterns shared several amplification products that allowed species identification. Importantly, the method was able to distinguish between species of the closely related Saccharomyces sensu stricto yeasts. In two cases where isolates could not be positively identified there was discrepancy between the phenetic and phylogenetic species concept. The method has applications in yeast ecological studies, enabling the rapid grouping of isolates with related genomes and the investigation of population dynamics of strains of the same species.

PCR differentiation of commercial yeast strains using intron splice site primers.

The increased use of pure starter cultures in the wine industry has made it necessary to develop a rapid and simple identification system for yeast strains. A method based upon the PCR using oligonucleotide primers that are complementary to intron splice sites has been developed. Since most introns are not essential for gene function, introns have evolved with minimal constraint. By targeting these highly variable sequences, the PCR has proved to be very effective in uncovering polymorphisms in commercial yeast strains. The speed of the method and the ability to analyze many samples in a single day permit the monitoring of specific yeast strains during fermentations. Furthermore, the simplicity of the technique, which does not require the isolation of DNA, makes it accessible to industrial laboratories that have limited molecular expertise and resources.

N-myristoylation is required for function of the pheromone-responsive G alpha protein of yeast: conditional activation of the pheromone response by a temperature-sensitive N-myristoyl transferase.

In a screen designed to identify novel mutations in the mating response pathway of Saccharomyces cerevisiae, we isolated conditional alleles of NMT1, the gene encoding N-myristoyl transferase. Genetic data indicate that Nmt1 deficiency results in the activation of the pheromone response at the level of Gpa1, the alpha subunit of the pheromone-responsive G protein. We show that Gpa1 is myristoylated by Nmt1, and without this normally stable modification, Gpa1 is unable to inhibit pheromone signaling. This loss of Gpa1 function is probably not the result of improper subcellular localization. Unlike the mammalian G alpha i proteins alpha i and alpha o, nonmyristoylated Gpa1 is able to associate with membranes. In addition to Gpa1, our data indicate that Nmt1 myristoylates other proteins essential to vegetative growth.

Mutations in cell division cycle genes CDC36 and CDC39 activate the Saccharomyces cerevisiae mating pheromone response pathway.

Conditional mutations in the genes CDC36 and CDC39 cause arrest in the G1 phase of the Saccharomyces cerevisiae cell cycle at the restrictive temperature. We present evidence that this arrest is a consequence of a mutational activation of the mating pheromone response. cdc36 and cdc39 mutants expressed pheromone-inducible genes in the absence of pheromone and conjugated in the absence of a mating pheromone receptor. On the other hand, cells lacking the G beta subunit or overproducing the G alpha subunit of the transducing G protein that couples the receptor to the pheromone response pathway prevented constitutive activation of the pathway in cdc36 and cdc39 mutants. These epistasis relationships imply that the CDC36 and CDC39 gene products act at the level of the transducing G protein. The CDC36 and CDC39 gene products have a role in cellular processes other than the mating pheromone response. A mating-type heterozygous diploid cell, homozygous for either the cdc36 or cdc39 mutation, does not exhibit the G1 arrest phenotype but arrests asynchronously with respect to the cell cycle. A similar asynchronous arrest was observed in cdc36 and cdc39 cells where the pheromone response pathway had been inactivated by mutations in the transducing G protein. Furthermore, cdc36 and cdc39 mutants, when grown on carbon catabolite-derepressing medium, did not arrest in G1 and did not induce pheromone-specific genes at the restrictive temperature.

A family of cyclin homologs that control the G1 phase in yeast.

Two Saccharomyces cerevisiae genes were isolated based upon their dosage-dependent rescue of a temperature-sensitive mutation of the gene CDC28, which encodes a protein kinase involved in control of cell division. CLN1 and CLN2 encode closely related proteins that also share homology with cyclins. Cyclins, characterized by a dramatic periodicity of abundance through the cell cycle, are thought to be involved in mitotic induction in animal cells. A dominant mutation in the CLN2 gene, CLN2-1, advances the G1- to S-phase transition in cycling cells and impairs the ability of cells to arrest in G1 phase in response to external signals, suggesting that the encoded protein is involved in G1 control of the cell cycle in Saccharomyces.